RESUMEN
Barmah Forest virus (BFV) is a mosquito-borne virus that causes arthralgia with accompanying rash, fever, and myalgia in humans. The virus is mainly found in Australia and has caused outbreaks associated with significant health concerns. As the sole representative of the Barmah Forest complex within the genus Alphavirus, BFV is not closely related genetically to other alphaviruses. Notably, basic knowledge of BFV molecular virology has not been well studied due to a lack of critical investigative tools such as an infectious clone. Here we describe the construction of an infectious BFV cDNA clone based on Genbank sequence and demonstrate that the clone-derived virus has in vitro and in vivo properties similar to naturally occurring virus, BFV field isolate 2193 (BFV2193-FI). A substitution in nsP4, V1911D, which was identified in the Genbank reference sequence, was found to inhibit virus rescue and replication. T1325P substitution in nsP2 selected during virus passaging was shown to be an adaptive mutation, compensating for the inhibitory effect of nsP4-V1911D. The two mutations were associated with changes in viral non-structural polyprotein processing and type I interferon (IFN) induction. Interestingly, a nuclear localization signal, active in mammalian but not mosquito cells, was identified in nsP3. A point mutation abolishing nsP3 nuclear localization attenuated BFV replication. This effect was more prominent in the presence of type I interferon signaling, suggesting nsP3 nuclear localization might be associated with IFN antagonism. Furthermore, abolishing nsP3 nuclear localization reduced virus replication in mice but did not significantly affect disease.IMPORTANCEBarmah Forest virus (BFV) is Australia's second most prevalent arbovirus, with approximately 1,000 cases reported annually. The clinical symptoms of BFV infection include rash, polyarthritis, arthralgia, and myalgia. As BFV is not closely related to other pathogenic alphaviruses or well-studied model viruses, our understanding of its molecular virology and mechanisms of pathogenesis is limited. There is also a lack of molecular tools essential for corresponding studies. Here we describe the construction of an infectious clone of BFV, variants harboring point mutations, and sequences encoding marker protein. In infected mammalian cells, nsP3 of BFV was located in the nuclei. This finding extends our understanding of the diverse mechanisms used by alphavirus replicase proteins to interact with host cells. Our novel observations highlight the complex synergy through which the viral replication machinery evolves to correct mutation errors within the viral genome.
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Infecciones por Alphavirus , Alphavirus , Genoma Viral , Proteínas no Estructurales Virales , Replicación Viral , Replicación Viral/genética , Proteínas no Estructurales Virales/genética , Proteínas no Estructurales Virales/metabolismo , Animales , Alphavirus/genética , Alphavirus/patogenicidad , Ratones , Infecciones por Alphavirus/virología , Genoma Viral/genética , Línea Celular , Humanos , AustraliaRESUMEN
With their unusually large genome and particle sizes, giant viruses (GVs) defy the conventional definition of viruses. Although most GVs isolated infect unicellular protozoans, such as amoeba, studies in the last decade have established their much wider prevalence infecting most eukaryotic supergroups and some giant viral families with the potential to be human pathogens. Their complexity, almost autonomous life cycle, and enigmatic evolution necessitate the study of GVs. The accurate assessment of GV proteome is a veritable challenge. We have compared the coverage of global protein identification using different methods for GVs isolated in Mumbai, Mimivirus Bombay (MVB), Powai Lake Megavirus (PLMV), and Kurlavirus (KV), along with two previously studied GVs, Acanthamoeba polyphaga Mimivirus (APMV) and Marseillevirus (MV). Our study shows that the simultaneous use of in-gel and in-solution digestion methods can significantly increase the coverage of protein identification in the global proteome analysis of purified GV particles. Combining the two methods of analyses, we identified an additional 72 proteins in APMV and 114 in MV compared with what have been previously reported. Similarly, proteomes of MVB, PLMV, and KV were analyzed, and a total of 242 proteins in MVB, 287 proteins in PLMV, and 174 proteins in KV were identified. Our results suggest that a combined methodology of in-gel and in-solution methods is more efficient and opens up new avenues for innovation in global proteome analysis of GVs. Future planetary health research on GVs can benefit from consideration of a broader range of proteomics methodologies as illustrated by the present study.
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Virus Gigantes , Proteoma , Proteómica , Proteómica/métodos , Virus Gigantes/genética , Virus Gigantes/metabolismo , Proteínas Virales/metabolismoRESUMEN
There is a growing appreciation that the direct interaction between bacteriophages and the mammalian host can facilitate diverse and unexplored symbioses. Yet the impact these bacteriophages may have on mammalian cellular and immunological processes is poorly understood. Here, we applied highly purified phage T4, free from bacterial by-products and endotoxins to mammalian cells and analyzed the cellular responses using luciferase reporter and antibody microarray assays. Phage preparations were applied in vitro to either A549 lung epithelial cells, MDCK-I kidney cells, or primary mouse bone marrow derived macrophages with the phage-free supernatant serving as a comparative control. Highly purified T4 phages were rapidly internalized by mammalian cells and accumulated within macropinosomes but did not activate the inflammatory DNA response TLR9 or cGAS-STING pathways. Following 8 hours of incubation with T4 phage, whole cell lysates were analyzed via antibody microarray that detected expression and phosphorylation levels of human signaling proteins. T4 phage application led to the activation of AKT-dependent pathways, resulting in an increase in cell metabolism, survival, and actin reorganization, the last being critical for macropinocytosis and potentially regulating a positive feedback loop to drive further phage internalization. T4 phages additionally down-regulated CDK1 and its downstream effectors, leading to an inhibition of cell cycle progression and an increase in cellular growth through a prolonged G1 phase. These interactions demonstrate that highly purified T4 phages do not activate DNA-mediated inflammatory pathways but do trigger protein phosphorylation cascades that promote cellular growth and survival. We conclude that mammalian cells are internalizing bacteriophages as a resource to promote cellular growth and metabolism.
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Anticuerpos , Bacteriófago T4 , Animales , Ratones , Humanos , Bacteriófago T4/genética , Ciclo Celular , ADN , Mamíferos/genéticaRESUMEN
Viruses are believed to be the obligate intracellular parasites that only carry genes essential for infecting and hijacking the host cell machinery. However, a recently discovered group of viruses belonging to the phylum nucleocytovirocota, also known as the nucleo-cytoplasmic large DNA viruses (NCLDVs), possess a number of genes that code for proteins predicted to be involved in metabolism, and DNA replication, and repair. In the present study, first, using proteomics of viral particles, we show that several proteins required for the completion of the DNA base excision repair (BER) pathway are packaged within the virions of Mimivirus as well as related viruses while they are absent from the virions of Marseillevirus and Kurlavirus that are NCLDVs with smaller genomes. We have thoroughly characterized three putative base excision repair enzymes from Mimivirus, a prototype NCLDV and successfully reconstituted the BER pathway using the purified recombinant proteins. The mimiviral uracil-DNA glycosylase (mvUDG) excises uracil from both ssDNA and dsDNA, a novel finding contrary to earlier studies. The putative AP-endonuclease (mvAPE) specifically cleaves at the abasic site created by the glycosylase while also exhibiting the 3'-5' exonuclease activity. The Mimivirus polymerase X protein (mvPolX) can bind to gapped DNA substrates and perform single nucleotide gap-filling followed by downstream strand displacement. Furthermore, we show that when reconstituted in vitro, mvUDG, mvAPE, and mvPolX function cohesively to repair a uracil-containing DNA predominantly by long patch BER and together, may participate in the BER pathway during the early phase of Mimivirus life-cycle.
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Reparación del ADN , ADN-(Sitio Apurínico o Apirimidínico) Liasa , Mimiviridae , ADN , Replicación del ADN , ADN-(Sitio Apurínico o Apirimidínico) Liasa/genética , Uracilo/metabolismo , Mimiviridae/genéticaRESUMEN
Viruses form extensive interfaces with host proteins to modulate the biology of the infected cell, frequently via multifunctional viral proteins. These proteins are conventionally considered as assemblies of independent functional modules, where the presence or absence of modules determines the overall composite phenotype. However, this model cannot account for functions observed in specific viral proteins. For example, rabies virus (RABV) P3 protein is a truncated form of the pathogenicity factor P protein, but displays a unique phenotype with functions not seen in longer isoforms, indicating that changes beyond the simple complement of functional modules define the functions of P3. Here, we report structural and cellular analyses of P3 derived from the pathogenic RABV strain Nishigahara (Nish) and an attenuated derivative strain (Ni-CE). We identify a network of intraprotomer interactions involving the globular C-terminal domain and intrinsically disordered regions (IDRs) of the N-terminal region that characterize the fully functional Nish P3 to fluctuate between open and closed states, whereas the defective Ni-CE P3 is predominantly open. This conformational difference appears to be due to the single mutation N226H in Ni-CE P3. We find that Nish P3, but not Ni-CE or N226H P3, undergoes liquid-liquid phase separation and this property correlates with the capacity of P3 to interact with different cellular membrane-less organelles, including those associated with immune evasion and pathogenesis. Our analyses propose that discrete functions of a critical multifunctional viral protein depend on the conformational arrangements of distant individual domains and IDRs, in addition to their independent functions.
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Virus de la Rabia , Rabia , Humanos , Virus de la Rabia/genética , Proteínas Virales/genética , Proteínas Virales/metabolismo , Factores de Virulencia/metabolismo , Isoformas de Proteínas/metabolismoRESUMEN
The nucleolus is a common target of viruses and viral proteins, but for many viruses the functional outcomes and significance of this targeting remains unresolved. Recently, the first intranucleolar function of a protein of a cytoplasmically-replicating negative-sense RNA virus (NSV) was identified, with the finding that the matrix (M) protein of Hendra virus (HeV) (genus Henipavirus, family Paramyxoviridae) interacts with Treacle protein within nucleolar subcompartments and mimics a cellular mechanism of the nucleolar DNA-damage response (DDR) to suppress ribosomal RNA (rRNA) synthesis. Whether other viruses utilise this mechanism has not been examined. We report that sub-nucleolar Treacle targeting and modulation is conserved between M proteins of multiple Henipaviruses, including Nipah virus and other potentially zoonotic viruses. Furthermore, this function is also evident for P3 protein of rabies virus, the prototype virus of a different RNA virus family (Rhabdoviridae), with Treacle depletion in cells also found to impact virus production. These data indicate that unrelated proteins of viruses from different families have independently developed nucleolar/Treacle targeting function, but that modulation of Treacle has distinct effects on infection. Thus, subversion of Treacle may be an important process in infection by diverse NSVs, and so could provide novel targets for antiviral approaches with broad specificity.
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Virus Hendra , Lyssavirus , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , ARN Ribosómico , Lyssavirus/genética , Lyssavirus/metabolismo , Ribosomas/metabolismo , Virus Hendra/genética , Virus Hendra/metabolismo , Factores de TranscripciónRESUMEN
Viral hijacking of microtubule (MT)-dependent transport is well understood, but several viruses also express discrete MT-associated proteins (vMAPs), potentially to modulate MT-dependent processes in the host cell. Specific roles for vMAP-MT interactions include subversion of antiviral responses by P3, an isoform of the P protein of rabies virus (RABV; genus Lyssavirus), which mediates MT-dependent antagonism of interferon (IFN)-dependent signal transducers and activators of transcription 1 (STAT1) signaling. P3 also undergoes nucleocytoplasmic trafficking and inhibits STAT1-DNA binding, indicative of intranuclear roles in a multipronged antagonistic strategy. MT association/STAT1 antagonist functions of P3 correlate with pathogenesis, indicating potential as therapeutic targets. However, key questions remain, including whether other P protein isoforms interact with MTs, the relationship of these interactions with pathogenesis, and the extent of conservation of P3-MT interactions between diverse pathogenic lyssaviruses. Using super-resolution microscopy, live-cell imaging, and immune signaling analyses, we find that multiple P protein isoforms associate with MTs and that association correlates with pathogenesis. Furthermore, P3 proteins from different lyssaviruses exhibit variation in intracellular localization phenotypes that are associated with STAT1 antagonist function, whereby P3-MT association is conserved among lyssaviruses of phylogroup I but not phylogroup II, while nucleocytoplasmic localization varies between P3 proteins of the same phylogroup within both phylogroup I and II. Nevertheless, the divergent P3 proteins retain significant IFN antagonist function, indicative of adaptation to favor different inhibitory mechanisms, with MT interaction important to phylogroup I viruses. IMPORTANCE Lyssaviruses, including rabies virus, cause rabies, a progressive encephalomyelitis that is almost invariably fatal. There are no effective antivirals for symptomatic infection, and effective application of current vaccines is limited in areas of endemicity, such that rabies causes ~59,000 deaths per year. Viral subversion of host cell functions, including antiviral immunity, is critical to disease, and isoforms of the lyssavirus P protein are central to the virus-host interface underpinning immune evasion. Here, we show that specific cellular interactions of P protein isoforms involved in immune evasion vary significantly between different lyssaviruses, indicative of distinct strategies to evade immune responses. These findings highlight the diversity of the virus-host interface, an important consideration in the development of pan-lyssavirus therapeutic approaches.
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Lyssavirus , Vacunas Antirrábicas , Virus de la Rabia , Rabia , Humanos , Lyssavirus/genética , Interferones/metabolismo , Virus de la Rabia/genética , Antivirales/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , ADN/metabolismoRESUMEN
Antagonism of the interferon (IFN)-mediated antiviral state is critical to infection by rabies virus (RABV) and other viruses, and involves interference in the IFN induction and signaling pathways in infected cells, as well as deactivation of the antiviral state in cells previously activated by IFN. The latter is required for viral spread in the host, but the precise mechanisms involved and roles in RABV pathogenesis are poorly defined. Here, we examined the capacity of attenuated and pathogenic strains of RABV that differ only in the IFN-antagonist P protein to overcome an established antiviral state. Importantly, P protein selectively targets IFN-activated phosphorylated STAT1 (pY-STAT1), providing a molecular tool to elucidate specific roles of pY-STAT1. We find that the extended antiviral state is dependent on a low level of pY-STAT1 that appears to persist at a steady state through ongoing phosphorylation/dephosphorylation cycles, following an initial IFN-induced peak. P protein of pathogenic RABV binds and progressively accumulates pY-STAT1 in inactive cytoplasmic complexes, enabling recovery of efficient viral replication over time. Thus, P protein-pY-STAT1 interaction contributes to 'disarming' of the antiviral state. P protein of the attenuated RABV is defective in this respect, such that replication remains suppressed over extended periods in cells pre-activated by IFN. These data provide new insights into the nature of the antiviral state, indicating key roles for residual pY-STAT1 signaling. They also elucidate mechanisms of viral deactivation of antiviral responses, including specialized functions of P protein in selective targeting and accumulation of pY-STAT1.
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Antivirales , Virus de la Rabia , Antivirales/farmacología , Interferones/metabolismo , Fosforilación , Virus de la Rabia/metabolismo , Factor de Transcripción STAT1/metabolismo , Replicación ViralRESUMEN
The rabies virus (RABV) phosphoprotein (P protein) is expressed as several isoforms, which differ in nucleocytoplasmic localization and microtubule (MT) association, mediated by several sequences, including nuclear localization (NLS) and export (NES) sequences. This appears to underpin a functional diversity enabling multiple functions in viral replication and modulation of host biology. Mechanisms regulating trafficking are poorly defined, but phosphorylation by protein kinase C (PKC) in the P protein C-terminal domain (PCTD) regulates nuclear trafficking, mediated by PCTD-localized NLS/NES sequences, indicating that phosphorylation contributes to functional diversity. The molecular mechanism underlying the effects of PKC, and potential roles in regulating other host-cell interactions are unresolved. Here, we assess effects of phosphorylation on the P3 isoform, which differs from longer isoforms through an ability to localize to the nucleus and associate with MTs, which are associated with antagonism of interferon (IFN) signaling. We find that phosphomimetic mutation of the PKC site S210 inhibits nuclear accumulation and MT association/bundling. Structural analysis indicated that phosphomimetic mutation induces no significant structural change to the NLS/NES but results in the side chain of N226 switching its interactions from E228, within the NES, to E210. Intriguingly, N226 is the sole substituted residue between the PCTD of the pathogenic IFN-resistant RABV strain Nishigahara and a derivative attenuated IFN-sensitive strain Ni-CE, inhibiting P3 nuclear localization and MT association. Thus, S210 phosphorylation appears to impact on N226/E228 to regulate P protein localization, with N226 mutation in Ni-CE mimicking a constitutively phosphorylated state resulting in IFN sensitivity and attenuation. IMPORTANCE Rabies virus P protein is a multifunctional protein with critical roles in replication and manipulation of host-cell processes, including subversion of immunity. This functional diversity involves interactions of several P protein isoforms with the cell nucleus and microtubules. Previous studies showed that phosphorylation of the P protein C-terminal domain (PCTD) at S210, near nuclear trafficking sequences, regulates nucleocytoplasmic localization, indicating key roles in functional diversity. The molecular mechanisms of this regulation have remained unknown. Here, we show that phosphomimetic mutation of S210 regulates nuclear localization and MT association. This regulation does not appear to result from disrupted PCTD structure, but rather from a switch of specific side chain interactions of N226. Intriguingly, N226 was previously implicated in P protein nuclear localization/MT association, immune evasion, and RABV pathogenesis, through undefined mechanisms. Our data indicate that the S210-N226 interface is a key regulator of virus-host interactions, which is significant for pathogenesis.
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Chaperonas Moleculares , Virus de la Rabia , Proteínas Estructurales Virales , Animales , Núcleo Celular/metabolismo , Fosforilación , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Virus de la Rabia/genética , Virus de la Rabia/metabolismoRESUMEN
BACKGROUND: The integrity of microtubule filament networks is essential for the roles in diverse cellular functions, and disruption of its structure or dynamics has been explored as a therapeutic approach to tackle diseases such as cancer. Microtubule-interacting drugs, sometimes referred to as antimitotics, are used in cancer therapy to target and disrupt microtubules. However, due to associated side effects on healthy cells, there is a need to develop safer drug regimens that still retain clinical efficacy. Currently, many questions remain open regarding the extent of effects on cellular physiology of microtubule-interacting drugs at clinically relevant and low doses. Here, we use super-resolution microscopies (single-molecule localization and optical fluctuation based) to reveal the initial microtubule dysfunctions caused by nanomolar concentrations of colcemid. RESULTS: We identify previously undetected microtubule (MT) damage caused by clinically relevant doses of colcemid. Short exposure to 30-80 nM colcemid results in aberrant microtubule curvature, with a trend of increased curvature associated to increased doses, and curvatures greater than 2 rad/µm, a value associated with MT breakage. Microtubule fragmentation was detected upon treatment with ≥ 100 nM colcemid. Remarkably, lower doses (< 20 nM after 5 h) led to subtle but significant microtubule architecture remodelling characterized by increased curvature and suppression of microtubule dynamics. CONCLUSIONS: Our results support the emerging hypothesis that microtubule-interacting drugs induce non-mitotic effects in cells, and establish a multi-modal imaging assay for detecting and measuring nanoscale microtubule dysfunction. The sub-diffraction visualization of these less severe precursor perturbations compared to the established antimitotic effects of microtubule-interacting drugs offers potential for improved understanding and design of anticancer agents.
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Citoesqueleto , Microtúbulos , Demecolcina/farmacología , Microscopía FluorescenteRESUMEN
[This corrects the article DOI: 10.1371/journal.ppat.1009800.].
RESUMEN
Although the majority of viruses of the family Mononegvirales replicate exclusively in the host cell cytoplasm, many of these viruses encode proteins that traffic between the nucleus and cytoplasm, which is believed to enable accessory functions in modulating the biology of the infected host cell. Among these, the P3 protein of rabies virus localizes to the nucleus through the activity of several specific nuclear localization and nuclear export signals. The major defined functions of P3 are in evasion of interferon (IFN)-mediated antiviral responses, including through inhibition of DNA-binding by IFN-activated STAT1. P3 also localizes to nucleoli and promyelocytic leukemia (PML) nuclear bodies, and interacts with nucleolin and PML protein, indicative of several intranuclear roles. The relationship of P3 nuclear localization with pathogenicity, however, is unresolved. We report that nucleocytoplasmic localization of P3 proteins from a pathogenic RABV strain, Nishigahara (Ni) and a non-pathogenic Ni-derived strain, Ni-CE, differs significantly, with nuclear accumulation defective for Ni-CE-P3. Molecular mapping indicates that altered localization derives from a coordinated effect, including two residue substitutions that independently disable nuclear localization and augment nuclear export signals, collectively promoting nuclear exclusion. Intriguingly, this appears to relate to effects on protein conformation or regulatory mechanisms, rather than direct modification of defined trafficking signal sequences. These data provide new insights into the role of regulated nuclear trafficking of a viral protein in the pathogenicity of a virus that replicates in the cytoplasm.
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Virus de la Rabia , Núcleo Celular/metabolismo , Señales de Exportación Nuclear , Virus de la Rabia/metabolismo , Proteínas Virales/metabolismo , VirulenciaRESUMEN
Type I Interferons (IFN-Is) are a family of cytokines which play a major role in inhibiting viral infection. Resultantly, many viruses have evolved mechanisms in which to evade the IFN-I response. Here we tested the impact of expression of 27 different SARS-CoV-2 genes in relation to their effect on IFN production and activity using three independent experimental methods. We identified six gene products; NSP6, ORF6, ORF7b, NSP1, NSP5 and NSP15, which strongly (>10-fold) blocked MAVS-induced (but not TRIF-induced) IFNß production. Expression of the first three of these SARS-CoV-2 genes specifically blocked MAVS-induced IFNß-promoter activity, whereas all six genes induced a collapse in IFNß mRNA levels, corresponding with suppressed IFNß protein secretion. Five of these six genes furthermore suppressed MAVS-induced activation of IFNλs, however with no effect on IFNα or IFNγ production. In sharp contrast, SARS-CoV-2 infected cells remained extremely sensitive to anti-viral activity exerted by added IFN-Is. None of the SARS-CoV-2 genes were able to block IFN-I signaling, as demonstrated by robust activation of Interferon Stimulated Genes (ISGs) by added interferon. This, despite the reduced levels of STAT1 and phospho-STAT1, was likely caused by broad translation inhibition mediated by NSP1. Finally, we found that a truncated ORF7b variant that has arisen from a mutant SARS-CoV-2 strain harboring a 382-nucleotide deletion associating with mild disease (Δ382 strain identified in Singapore & Taiwan in 2020) lost its ability to suppress type I and type III IFN production. In summary, our findings support a multi-gene process in which SARS-CoV-2 blocks IFN-production, with ORF7b as a major player, presumably facilitating evasion of host detection during early infection. However, SARS-CoV-2 fails to suppress IFN-I signaling thus providing an opportunity to exploit IFN-Is as potential therapeutic antiviral drugs.
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Interferón beta/metabolismo , SARS-CoV-2/inmunología , Proteínas Virales/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Animales , Chlorocebus aethiops , Factor 2 Eucariótico de Iniciación/metabolismo , Células HEK293 , Humanos , Interferón beta/genética , Interferón beta/farmacología , SARS-CoV-2/efectos de los fármacos , Factor de Transcripción STAT1/metabolismo , Células Vero , Proteínas Virales/genéticaRESUMEN
Viral interferon (IFN) antagonist proteins mediate evasion of IFN-mediated innate immunity and are often multifunctional, with distinct roles in viral replication. The Ebola virus IFN antagonist VP24 mediates nucleocapsid assembly, and inhibits IFN-activated signaling by preventing nuclear import of STAT1 via competitive binding to nuclear import receptors (karyopherins). Proteins of many viruses, including viruses with cytoplasmic replication cycles, interact with nuclear trafficking machinery to undergo nucleocytoplasmic transport, with key roles in pathogenesis; however, despite established karyopherin interaction, potential nuclear trafficking of VP24 has not been investigated. We find that inhibition of nuclear export pathways or overexpression of VP24-binding karyopherin results in nuclear localization of VP24. Molecular mapping indicates that cytoplasmic localization of VP24 depends on a CRM1-dependent nuclear export sequence at the VP24 C-terminus. Nuclear export is not required for STAT1 antagonism, consistent with competitive karyopherin binding being the principal antagonistic mechanism, while export mediates return of nuclear VP24 to the cytoplasm where replication/nucleocapsid assembly occurs.
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Núcleo Celular/virología , Citoplasma/virología , Ebolavirus/metabolismo , Fiebre Hemorrágica Ebola/virología , Interferón Tipo I/metabolismo , Proteínas Virales/metabolismo , Transporte Activo de Núcleo Celular , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Ebolavirus/química , Ebolavirus/genética , Fiebre Hemorrágica Ebola/genética , Fiebre Hemorrágica Ebola/metabolismo , Interacciones Huésped-Patógeno , Humanos , Interferón Tipo I/genética , Señales de Localización Nuclear , Factor de Transcripción STAT1/genética , Factor de Transcripción STAT1/metabolismo , Proteínas Virales/química , Proteínas Virales/genéticaRESUMEN
Rabies virus phosphoprotein (P protein) is a multifunctional protein that plays key roles in replication as the polymerase cofactor that binds to the complex of viral genomic RNA and the nucleoprotein (N protein), and in evading the innate immune response by binding to STAT transcription factors. These interactions are mediated by the C-terminal domain of P (PCTD). The colocation of these binding sites in the small globular PCTD raises the question of how these interactions underlying replication and immune evasion, central to viral infection, are coordinated and, potentially, coregulated. While direct data on the binding interface of the PCTD for STAT1 is available, the lack of direct structural data on the sites that bind N protein limits our understanding of this interaction hub. The PCTD was proposed to bind via two sites to a flexible loop of N protein (Npep) that is not visible in crystal structures, but no direct analysis of this interaction has been reported. Here we use Nuclear Magnetic Resonance, and molecular modelling to show N protein residues, Leu381, Asp383, Asp384 and phosphor-Ser389, are likely to bind to a 'positive patch' of the PCTD formed by Lys211, Lys214 and Arg260. Furthermore, in contrast to previous predictions we identify a single site of interaction on the PCTD by this Npep. Intriguingly, this site is proximal to the defined STAT1 binding site that includes Ile201 to Phe209. However, cell-based assays indicate that STAT1 and N protein do not compete for P protein. Thus, it appears that interactions critical to replication and immune evasion can occur simultaneously with the same molecules of P protein so that the binding of P protein to activated STAT1 can potentially occur without interrupting interactions involved in replication. These data suggest that replication complexes might be directly involved in STAT1 antagonism.
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Evasión Inmune/fisiología , Chaperonas Moleculares/metabolismo , Virus de la Rabia/metabolismo , Rabia/virología , Proteínas Estructurales Virales/metabolismo , Replicación Viral/fisiología , Animales , Células COS , Chlorocebus aethiops , Células HEK293 , Humanos , Proteínas de la Nucleocápside/metabolismo , Rabia/metabolismo , Factor de Transcripción STAT1/metabolismoRESUMEN
Many viruses target signal transducers and activators of transcription (STAT) 1 and 2 to antagonise antiviral interferon signalling, but targeting of signalling by other STATs/cytokines, including STAT3/interleukin 6 that regulate processes important to Ebola virus (EBOV) haemorrhagic fever, is poorly defined. We report that EBOV potently inhibits STAT3 responses to interleukin-6 family cytokines, and that this is mediated by the interferon-antagonist VP24. Mechanistic analysis indicates that VP24 effects a unique strategy combining distinct karyopherin-dependent and karyopherin-independent mechanisms to antagonise STAT3-STAT1 heterodimers and STAT3 homodimers, respectively. This appears to reflect distinct mechanisms of nuclear trafficking of the STAT3 complexes, revealed for the first time by our analysis of VP24 function. These findings are consistent with major roles for global inhibition of STAT3 signalling in EBOV infection, and provide new insights into the molecular mechanisms of STAT3 nuclear trafficking, significant to pathogen-host interactions, cell physiology and pathologies such as cancer.
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Fiebre Hemorrágica Ebola/metabolismo , Fiebre Hemorrágica Ebola/virología , Factor de Transcripción STAT3/antagonistas & inhibidores , Transducción de Señal/fisiología , Proteínas Virales/metabolismo , Animales , Chlorocebus aethiops , Ebolavirus , Células HEK293 , Humanos , Células VeroRESUMEN
Bats are reservoirs of many pathogenic viruses, including the lyssaviruses rabies virus (RABV) and Australian bat lyssavirus (ABLV). Lyssavirus strains are closely associated with particular host reservoir species, with evidence of specific adaptation. Associated phenotypic changes remain poorly understood but are likely to involve phosphoprotein (P protein), a key mediator of the intracellular virus-host interface. Here, we examine the phenotype of P protein of ABLV, which circulates as two defined lineages associated with frugivorous and insectivorous bats, providing the opportunity to compare proteins of viruses adapted to divergent bat species. We report that key functions of P protein in the antagonism of interferon/signal transducers and activators of transcription 1 (STAT1) signaling and the capacity of P protein to undergo nuclear trafficking differ between lineages. Molecular mapping indicates that these differences are functionally distinct and appear to involve modulatory effects on regulatory regions or structural impact rather than changes to defined interaction sequences. This results in partial but significant phenotypic divergence, consistent with "fine-tuning" to host biology, and with potentially distinct properties in the virus-host interface between bat families that represent key zoonotic reservoirs.
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Biodiversidad , Quirópteros/virología , Lyssavirus/fisiología , Fenotipo , Secuencia de Aminoácidos , Animales , Reservorios de Enfermedades , Interacciones Huésped-Patógeno , Interferones/metabolismo , Lyssavirus/clasificación , Factor de Transcripción STAT1/metabolismo , Transducción de Señal , Proteínas Virales/química , Proteínas Virales/genética , Proteínas Virales/metabolismoRESUMEN
Many viruses target signal transducer and activator of transcription (STAT) 1 to antagonise antiviral interferon signalling, but targeting of STAT3, a pleiotropic molecule that mediates signalling by diverse cytokines, is poorly understood. Here, using lyssavirus infection, quantitative live cell imaging, innate immune signalling and protein interaction assays, and complementation/depletion of STAT expression, we show that STAT3 antagonism is conserved among P-proteins of diverse pathogenic lyssaviruses and correlates with pathogenesis. Importantly, P-protein targeting of STAT3 involves a highly selective mechanism whereby P-protein antagonises cytokine-activated STAT3-STAT1 heterodimers, but not STAT3 homodimers. RT-qPCR and reporter gene assays indicate that this results in specific modulation of interleukin-6-dependent pathways, effecting differential antagonism of target genes. These data provide novel insights into mechanisms by which viruses can modulate cellular function to support infection through discriminatory targeting of immune signalling complexes. The findings also highlight the potential application of selective interferon-antagonists as tools to delineate signalling by particular STAT complexes, significant not only to pathogen-host interactions but also cell physiology, development and cancer.
Asunto(s)
Citocinas/metabolismo , Regulación de la Expresión Génica , Lyssavirus/inmunología , Infecciones por Rhabdoviridae/inmunología , Factor de Transcripción STAT1/metabolismo , Factor de Transcripción STAT3/metabolismo , Proteínas Virales/metabolismo , Células HEK293 , Células HeLa , Humanos , Interleucina-6/metabolismo , Infecciones por Rhabdoviridae/metabolismo , Infecciones por Rhabdoviridae/virología , Factor de Transcripción STAT1/genética , Factor de Transcripción STAT3/genética , Transactivadores , Proteínas Virales/genéticaRESUMEN
Viruses commonly antagonize the antiviral type I interferon response by targeting signal transducer and activator of transcription 1 (STAT1) and STAT2, key mediators of interferon signaling. Other STAT family members mediate signaling by diverse cytokines important to infection, but their relationship with viruses is more complex. Importantly, virus-STAT interaction can be antagonistic or stimulatory depending on diverse viral and cellular factors. While STAT antagonism can suppress immune pathways, many viruses promote activation of specific STATs to support viral gene expression and/or produce cellular conditions conducive to infection. It is also becoming increasingly clear that viruses can hijack noncanonical STAT functions to benefit infection. For a number of viruses, STAT function is dynamically modulated through infection as requirements for replication change. Given the critical role of STATs in infection by diverse viruses, the virus-STAT interface is an attractive target for the development of antivirals and live-attenuated viral vaccines. Here, we review current understanding of the complex and dynamic virus-STAT interface and discuss how this relationship might be harnessed for medical applications.
Asunto(s)
Factores de Transcripción STAT/metabolismo , Transducción de Señal/fisiología , Virus/metabolismo , Citocinas , Expresión Génica , Interacciones Huésped-Patógeno , Evasión Inmune , Factores de Transcripción STAT/genética , Factor de Transcripción STAT1/metabolismo , Factor de Transcripción STAT2/metabolismo , Factor de Transcripción STAT4 , Factor de Transcripción STAT6 , Virus/genéticaRESUMEN
Lyssavirus P protein is a multifunctional protein that interacts with numerous host-cell proteins. The C-terminal domain (CTD) of P is important for inhibition of JAK-STAT signaling enabling the virus to evade host immunity. Several regions on the surface of rabies virus P are reported to interact with host factors. Among them, an extended, discrete hydrophobic patch of P CTD is notable. Although structures of P CTD of two strains of rabies virus, and of mokola virus have been solved, the structure of P CTD for Duvenhage virus, which is functionally divergent from these species for immune evasion function, is not known. Here, we analyze the structures of P CTD of Duvenhage and of a distinct rabies virus strain to gain further insight on the nature and potential function of the hydrophobic surface. Molecular contacts in crystals suggest that the hydrophobic patch is important to intermolecular interactions with other proteins, which differ between the lyssavirus species.